P450 enzyme, enzyme composition, nucleic acid, expression vector, bioengineering strain, method and application

Abstract

The invention discloses a P450 enzyme, an enzyme composition, nucleic acid, an expression vector, a bioengineering strain, a method and application. The amino acid sequence of the P450 enzyme is as shown in SEQ ID NO. 1. According to the invention, the P450 enzyme capable of modifying bufalin and / or resibufogenin at a fixed point under a mild condition is found for the first time, and a bioengineering strain and a biocatalytic synthesis method are developed based on the P450 enzyme, so that a biosynthesis method for efficiently synthesizing bufadienolide compounds is established.

Description

Technical Field

[0001] The present invention relates to the field of biosynthesis, and in particular to a P450 enzyme and enzyme composition, nucleic acid, expression vector, bioengineering strain, method and application. Background Art

[0002] Bufalin, Resibufogenin and its modified derivatives are bufadienolide compounds, which are a class of compounds that can act on Na + / K + -ATPase and multiple cell signaling pathways, and steroid compounds with cardiotonic and anti-tumor activities. More than 100 bufadienolide compounds have been reported, all of which are derived from the parotid gland and skin secretions of toads. Their common structural characteristics are as follows:

[0003]

[0004] The AB and CD rings are cis-fused, while the BC ring is trans-fused. The 5β and 14β chemical bonds are crucial for maintaining the compound's stereoconfiguration, and C17 connects to the six-membered unsaturated lactone ring. Hydroxylation and further modifications at various positions on the steroid nucleus can affect the compound's activity.

[0005] According to literature reports, among bufadienolide compounds, 19-hydroxybufalin can inhibit the growth of liver, colon, and lung cancer cells, exhibiting strong tumor cytotoxicity. Furthermore, in nude mouse models of non-small cell lung cancer xenografts, it can reduce tumor size and weight without affecting normal mouse organs. Furthermore, 19-hydroxybufalin has anti-Chagas disease activity and is a potential drug for the treatment of Chagas disease. Furthermore, 32 of the isolated and reported bufadienolide compounds have a 19-hydroxyl modification or can be converted from 19-hydroxybufalin. According to literature reports, 1β-hydroxybufalin has strong cytotoxicity against a variety of tumor cells, including leukemia, liver cancer, breast cancer, colon cancer, lung cancer, prostate cancer, nasopharyngeal cancer, glioma, and pancreatic cancer, making it a potential anti-tumor drug.

[0006] While the aforementioned bufadienolide compounds possess diverse and highly effective pharmacological activities, their primary extraction and isolation from toad skin and parotid gland secretions presents challenges such as low concentrations, difficulty in extraction, and limited availability. Chemical methods also suffer from stereoselectivity, complex pathways, difficulty in separation, high costs, and environmental pollution. This severely limits the development and application of these active compounds. Summary of the Invention

[0007] To solve the problems existing in the above-mentioned prior art, the present invention provides a P450 enzyme and enzyme composition, nucleic acid, expression vector, bioengineering strain, method and application.

[0008] Specifically, the present invention provides:

[0009] (1) A P450 enzyme, characterized in that the amino acid sequence of the P450 enzyme is shown in SEQ ID NO.1.

[0010] (2) A nucleic acid encoding the P450 enzyme according to (1).

[0011] (3) The nucleic acid according to (2), wherein the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO. 2.

[0012] (4) An expression vector, characterized in that the expression vector contains a nucleic acid encoding the P450 enzyme according to (1).

[0013] (5) The expression vector according to (4), wherein the expression vector comprises the nucleotide sequence shown in SEQ ID NO.2.

[0014] (6) The expression vector according to (4), wherein the expression vector comprises a constitutive promoter HXT7p upstream of the nucleic acid encoding the P450 enzyme according to (1); preferably, the backbone of the expression vector is pRS426 or pRS425.

[0015] (7) An enzyme composition comprising the P450 enzyme according to (1) and cytochrome P450 enzyme reductase.

[0016] (8) The enzyme composition according to (7), wherein the cytochrome P450 enzyme reductase is selected from the group consisting of Ncp1, ATR1, AnCPR, TaCPR, RnCPR, XlCPR and BufoCPR.

[0017] (9) A bioengineering strain, characterized in that the bioengineering strain expresses the P450 enzyme according to (1); preferably, the bioengineering strain also expresses a cytochrome P450 enzyme reductase; preferably, the cytochrome P450 enzyme reductase is selected from Ncp1, ATR1, AnCPR, TaCPR, RnCPR, XlCPR and BufoCPR.

[0018] (10) The bioengineered strain according to (9), wherein the bioengineered strain is selected from Saccharomyces cerevisiae strains BY4742, BJ5464-NpgA, YRC01 and WAT11U.

[0019] (11) A method for preparing a bufadienolide compound, comprising contacting the P450 enzyme according to (1) with bufalin and / or resifugin in the presence of an electron donor, thereby performing a catalytic reaction to produce a compound represented by Formula 1, 2, 3 or 4 or a mixture thereof:

[0020]

[0021] (12) The method according to (11), wherein the electron donor is a cytochrome P450 enzyme reductase; preferably, the cytochrome P450 enzyme reductase is selected from Ncp1, ATR1, AnCPR, TaCPR, RnCPR, XlCPR and BufoCPR.

[0022] (13) The method according to (11), wherein the catalytic reaction is carried out at 28-30°C for 1-2 days; preferably, the catalytic reaction is carried out at 30°C for 1 day.

[0023] (14) The method according to (11), wherein the P450 enzyme according to (1) is contacted with bufatoxin to perform a catalytic reaction to generate the compounds shown in Formula 1 and Formula 2; or the P450 enzyme according to (1) is contacted with bufatoxin to perform a catalytic reaction to generate the compounds shown in Formula 3 and Formula 4.

[0024] (15) The method according to (11), wherein the method comprises the following steps:

[0025] 1) Providing the bioengineered strain according to (9) or (10);

[0026] 2) mixing the bioengineered strain with bufalin and / or resifugin to obtain a mixed culture, and incubating the mixed culture; preferably, the mixed culture further comprises 5-aminolevulinic acid hydrochloride.

[0027] (16) The method according to (15), wherein in step 2), the OD of the bioengineered strain is 600 The final concentration of bufalin and / or resifugin in the mixed culture is 200 μM to 1 mM, and the final concentration of 5-aminolevulinic acid hydrochloride in the mixed culture is 0.5-1 mM.

[0028] (17) The method according to (15), wherein the mixed incubation is carried out at 28-30°C and 220-250 rpm for 1-2 days; preferably, the mixed incubation is carried out at 30°C and 220 rpm for 1 day.

[0029] (18) The method according to (11), wherein the method further comprises purifying the compound represented by Formula 1, 2, 3 or 4 or a mixture thereof from the system obtained by the catalytic reaction.

[0030] (19) Use of the P450 enzyme described in (1), the nucleic acid described in (2) or (3), the expression vector described in any one of (4) to (6), the enzyme composition described in (7) or (8), or the bioengineering strain described in (9) or (10) in preparing the compound represented by formula 1, 2, 3 or 4 or a mixture thereof.

[0031] (20) A bufadienolide compound, which is obtained by catalyzing a reaction of the P450 enzyme according to (1) with lipobufogenin as a substrate in the presence of an electron donor; wherein the structural formula of the bufadienolide compound is shown in Formula 4:

[0032]

[0033] Compared with the prior art, the present invention has the following advantages and positive effects:

[0034] 1. The present invention discovered for the first time the P450 enzyme CYP46A35 that can modify bufalin and resifobafoxin. It can biocatalyze bufalin to produce 19-hydroxybufalin and 1β-hydroxybufalin, or catalyze resifobafoxin to produce 1β-hydroxyresifobafoxin and 2β-hydroxyresifobafoxin under mild conditions, thereby providing a new site-specific modification and efficient biosynthesis method, thereby realizing the simple and efficient acquisition of these bufadienolide compounds.

[0035] 2. The present invention takes into account that the P450 enzyme of eukaryotic origin is a membrane protein located in the endoplasmic reticulum. Therefore, Saccharomyces cerevisiae, which has an endoplasmic reticulum organelle, is used to express the P450 enzyme CYP46A35 from toads. The expression effect is ideal, and the expressed enzyme has the desired biological activity. In addition, the yeast used grows rapidly, is simple to culture, has a short conversion cycle, and is low in cost, thereby enabling the low-cost, simple and efficient synthesis of specific bufadienolide compounds.

[0036] 3. The present invention screened different yeast chassis strains and cytochrome P450 reductase (CPR), a key protein involved in electron transfer. It was found that different yeast chassis strains and different CPR and CYP46A35 combinations resulted in different electron transfer efficiencies and catalytic activities. Thus, through screening and optimization, the present invention achieved an approximately 6-fold increase in substrate conversion efficiency.

[0037] 4. The P450 enzymes of the present invention provide new candidate enzymes for the synthesis of bufadienolide compounds; the bioengineered strains and biocatalytic synthesis methods of the present invention provide new biocatalytic pathways for the synthesis of bufadienolide compounds. The present invention identifies enzymes that can catalyze the conversion of bufalin or resibufogenin to produce active bufadienolide compounds, and synthesizes them using biocatalysis, laying the foundation for the development and application of such compounds.

[0038] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 A schematic diagram of a catalytic reaction according to one embodiment of the present invention is shown.

[0040] Figure 2 A schematic diagram of a catalytic reaction according to one embodiment of the present invention is shown.

[0041] Figure 3 The HPLC chromatograms of the catalytic reaction using the bioengineered strain of the present invention in Example 3 of the present application are shown. i) is the HPLC curve of the standard bufalin; ii) is the HPLC curve of the product obtained by the catalytic reaction using bioengineered strain B (WAT11U (RnCPR)); and iii) is the HPLC curve of the product obtained by the catalytic reaction using bioengineered strain A (WAT11U (RnCPR+CYP46A35)).

[0042] Figure 4 The following HPLC chromatograms are obtained from the catalytic reaction using the bioengineered strains of the present invention in Example 4 of the present application. i) is the HPLC curve of the standard resifugin; ii) is the HPLC curve of the product obtained by the catalytic reaction using bioengineered strain B (WAT11U (RnCPR)); and iii) is the HPLC curve of the product obtained by the catalytic reaction using bioengineered strain A (WAT11U (RnCPR+CYP46A35)).

[0043] Figure 5 Shown are high-resolution mass spectra of the products in Example 5 of the present application.

[0044] Figure 6 The NMR spectrum of the isolated product 2β-hydroxy lipobufogenin (dissolved in deuterated methanol) is shown. 1 H NMR spectrum (600MHz); Figure B is 13 C NMR spectrum (150MHz); Figure C is 1 H- 1Figure D is the HSQC NMR spectrum; Figure E is the HMBC NMR spectrum; and Figure F is the NOSEY NMR spectrum. The structural formula of the product, 2β-hydroxylipolytoxin, is shown below:

[0045] DETAILED DESCRIPTION

[0046] The following describes several preferred embodiments of the present invention with reference to the accompanying drawings to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0047] It should be understood that the above-mentioned technical features of the present invention and the technical features specifically described below (including but not limited to implementation plans and examples) can be combined with each other in any appropriate manner to form a new or preferred technical solution, as long as there is no contradiction and the combined technical solution can be successfully implemented and can solve the technical problem of the present invention. The any appropriate manner shall be based on the ability to implement the technical solution of the present invention, solve the technical problem of the present invention, and achieve the corresponding technical effect.

[0048] In the present invention, words such as “including”, “further”, “having”, and “further” merely indicate better or more specific implementation methods or examples, and should be understood that they do not constitute a limitation on the scope of protection of the present invention.

[0049] In the present invention, "and / or" means any one or any combination of the listed items.

[0050] Unless otherwise specified, the numerical ranges in the present invention include both endpoints.

[0051] According to one aspect of the present invention, a P450 enzyme, CYP46A35, is a newly discovered P450 enzyme from the Chinese toad Bufo bufo gargarizans. It is capable of efficiently catalyzing the hydroxylation of inert C-H bonds at specific sites in bufalin and resifugin. The P450 enzyme's catalytic substrates are selected from bufalin and resifugin, and the bufadienolide compound produced by the catalytic reaction is selected from the compounds represented by the following formulas 1, 2, 3, and 4:

[0052]

[0053]

[0054] In one embodiment of the present invention, Figure 1As shown, the P450 enzyme CYP46A35 can catalyze the hydroxylation of the inert CH bonds at positions 1 and 19 of bufalin to generate compounds shown in Formula 1 and Formula 2.

[0055] In one embodiment of the present invention, Figure 2 As shown, the P450 enzyme CYP46A35 can catalyze the hydroxylation of the inert CH bonds at positions 1 and 2 of the resifugin to generate compounds shown in Formula 3 and Formula 4.

[0056] In one embodiment of the present invention, the P450 enzyme CYP46A35 can catalyze the hydroxylation of inert CH bonds at specific sites of bufalin and resifugin to produce compounds of Formulas 1, 2, 3, and 4.

[0057] The amino acid sequence of the P450 enzyme according to the present invention is shown in SEQ ID NO.1.

[0058] SEQ ID NO.1:

[0059] MELLGLVSWLLLLLLLTLVVTCFLLYCGYIHYQHMKYDHIPGPPRESFLFGHTSAIWKVMRKNQLVYDLFLNWVETYGPVIRINALHKVTILSVSPESVKDVLMSPKYRKDWFYDNLHTLFGVRLMG NGLVTDRDNDHWYKQRRIMDPAFSRTYLIGLLGPFNEKAEELMERLAEEADGRSHVVMHTMMSRVTLDVISKVAFGMETNSLKDDGTPLPRAISLVMRAFVEMRDPFIRYSREKQAFIRDVQESIRL LRKTGRECIERRQKAIQDGEEIPVDILTQILKGAALEGDCDMEDLLDNFVTFFIAGQETTANQLAFTIMELARNPKILEKTQAEVDEVIGVKRDIEYNDLGKLQYLSQVLKESLRLYPTAPGTSRE IEEETIIEGFRIPPKVNLMFNSYIMGRMQQLYPDPLTFNPERFHPDAPKPYYSYFPFSLGPRSCIGQVFAQMEAKVIMAKLLQRFQFELVEGQSFGIIDTASLRPKSGVICRLTIRTNPGEGKKVD*

[0060] The present invention also provides a nucleic acid encoding the P450 enzyme of the present invention. The nucleic acid can be DNA or RNA.

[0061] In some embodiments of the present invention, the nucleic acid comprises a nucleotide sequence encoding the P450 enzyme CYP46A35 of the present invention. Preferably, the nucleotide sequence is as shown in SEQ ID NO. 2.

[0062] SEQ ID NO.2:

[0063]

[0064] According to another aspect of the present invention, an expression vector is provided, which comprises the nucleic acid of the present invention to encode the P450 enzyme of the present invention.

[0065] Preferably, in the expression vector, the present invention utilizes the constitutive strong promoter HXT7p to enhance the expression of the P450 enzyme CYP46A35. In some preferred embodiments, the constitutive strong promoter HXT7p is located upstream of the nucleotide sequence encoding the P450 enzyme CYP46A35.

[0066] The backbone of the expression vector can be pRS426 or pRS425. According to some specific embodiments of the present invention, the backbone of the vector can be modified to be more suitable for expressing the enzyme of the present invention by inserting a specific promoter and terminator. For example, pRS426 is constructed as pRS426-HXT7p-FBA1t (the vector carries Ura3 for screening positive clones and retaining plasmids) by inserting the strong constitutive promoter HXT7p and terminator FBA1t.

[0067] In some preferred embodiments, the present invention also constructs an expression vector comprising a cytochrome P450 enzyme reductase. Preferably, the cytochrome P450 enzyme reductase is selected from Ncp1, ATR1, AnCPR, TaCPR, RnCPR, XlCPR, and BufoCPR. These cytochrome P450 enzyme reductases can provide electron donors for the biocatalysis of the P450 enzyme CYP46A35.

[0068] Preferably, the cytochrome P450 enzyme reductase Ncp1 is derived from Saccharomyces cerevisiae, ATR1 is derived from Arabidopsis thaliana, AnCPR is derived from Aspergillus nidulans, TaCPR is derived from Trichoderma asperellum, RnCPR is derived from Rattus norvegicus, XlCPR is derived from Xenopus laevis, and BufoCPR is derived from Bufo bufo gargarizans.

[0069] Preferably, the amino acid sequences of the cytochrome P450 enzyme reductase Ncp1, ATR1, AnCPR, TaCPR, RnCPR, XlCPR and BufoCPR are shown as SEQ ID NOs. 3-9, respectively.

[0070] The nucleotide sequences encoding the above cytochrome P450 enzyme reductase are shown in SEQ ID NOs. 10-16 respectively.

[0071] The backbone of the expression vector containing the cytochrome P450 enzyme reductase can be pRS426 or pRS425. According to some specific embodiments of the present invention, the backbone of the vector can be modified to be more suitable for expressing the cytochrome P450 enzyme reductase by inserting a specific promoter and terminator. For example, pRS425 is constructed as pRS425-TEF1p-PGK1t by inserting the constitutive promoter TEF1p and the terminator PGK1t (the vector comes with Leu2 for screening positive clones and retaining plasmids).

[0072] According to another aspect of the present invention, an enzyme composition is provided, comprising the P450 enzyme of the present invention and cytochrome P450 enzyme reductase.

[0073] Preferably, the cytochrome P450 enzyme reductase is selected from the above-mentioned Ncp1, ATR1, AnCPR, TaCPR, RnCPR, XlCPR and BufoCPR.

[0074] The P450 enzyme CYP46A35 of the present invention can efficiently synthesize the specific bufadienolide compound represented by formula 1-4 with cytochrome P450 enzyme reductase as an electron donor.

[0075] According to another aspect of the present invention, a bioengineering strain is provided, which expresses the P450 enzyme CYP46A35 of the present invention.

[0076] Preferably, the bioengineered strain further expresses a cytochrome P450 enzyme reductase. Preferably, the cytochrome P450 enzyme reductase is selected from the group consisting of Ncp1, ATR1, AnCPR, TaCPR, RnCPR, XlCPR, and BufoCPR.

[0077] The bioengineering strain can be made to express the P450 enzyme CYP46A35 and / or cytochrome P450 enzyme reductase by comprising the expression vector of the present invention. For example, the expression vector of the present invention can be transformed into the bioengineering strain to make it comprise the expression vector of the present invention.

[0078] In some specific embodiments, the bioengineered strains of the present invention can be constructed by the following methods:

[0079] Step (1): obtaining the P450 enzyme CYP46A35 gene from Bufo bufo gargarizans; obtaining the cytochrome P450 enzyme reductase (CPR) gene;

[0080] Step (2): cloning the P450 enzyme CYP46A35 gene and the CPR gene obtained in step (1) into expression vectors respectively, and screening positive plasmids;

[0081] Step (3): The positive plasmids obtained in step (2) are respectively transferred into host bacteria, thereby constructing the bioengineered strain of the present invention.

[0082] In some embodiments of the present invention, the cytochrome P450 enzyme reductase (CPR) gene in the above step (1) can be selected from Ncp1 obtained by genome amplification of Saccharomyces cerevisiae strain BY4742, ATR1 from Arabidopsis thaliana, AnCPR from Aspergillus nidulans, TaCPR from Trichoderma asperellum, RnCPR from Rattus norvegicus, XlCPR from Xenopus laevis, and BufoCPR from Bufobufo gargarizans.

[0083] The host bacteria in the above step (3) can be selected from Saccharomyces cerevisiae strains BY4742, BJ5464-NpgA, YRC01 and WAT11U.

[0084] According to a preferred embodiment of the present invention, the bioengineered strain of the present invention can be constructed by the following method:

[0085] Step (1): using the cDNA of the postauricular gland tissue of Bufo bufo gargarizans as a template, the P450 enzyme CYP46A35 gene was obtained by PCR amplification; using the genome of the yeast Saccharomyces cerevisiae strain BY4742 as a template, the cytochrome P450 enzyme reductase CPR gene ncp1 was obtained by PCR amplification;

[0086] Step (2): cloning the CYP46A35 gene and the CPR gene ncp1 obtained in step (1) into yeast expression vectors, respectively, and screening positive plasmids;

[0087] Step (3): The positive plasmids obtained in step (2) are respectively transferred into host bacteria, thereby constructing the bioengineered strain of the present invention.

[0088] In some embodiments, the bioengineered strain of the present invention can be cultured by the following method, which comprises: inoculating the bioengineered strain into SD-Ura-Leu medium and culturing until OD 600 The OD value was about 2, and then 2% (V / V) was transferred into SD-Ura-Leu medium and cultured for 2 days. 600 It is about 4.5.

[0089] According to another aspect of the present invention, a method for preparing a bufadienolide compound is provided, wherein the P450 enzyme of the present invention is contacted with a substrate in the presence of an electron donor, thereby performing a catalytic reaction to produce a bufadienolide compound. The substrate is selected from bufalin and resibufogenin, and the bufadienolide compound is selected from the compounds represented by the following formulas 1, 2, 3, or 4, or mixtures thereof:

[0090]

[0091] In some embodiments of the present invention, the P450 enzyme is contacted with the substrate bufalin in the presence of an electron donor to catalyze the hydroxylation of the inert CH bonds at positions 1 and 19 of bufalin, thereby generating compounds of Formula 1 and Formula 2.

[0092] In other embodiments of the present invention, the P450 enzyme is contacted with the substrate resibufogenin in the presence of an electron donor to catalyze the hydroxylation of the inert CH bonds at positions 1 and 2 of resibufogenin, thereby generating compounds shown in Formula 3 and Formula 4.

[0093] In some further embodiments of the present invention, the P450 enzyme is contacted with the substrates bufalin and resifugin in the presence of an electron donor to catalyze the hydroxylation of inert CH bonds at specific sites of bufalin and resifugin, thereby generating compounds shown in Formulas 1, 2, 3 and 4.

[0094] The desired product can be separated and purified from the catalytic reaction product by, for example, preparative reverse phase column chromatography, for example, separation and purification of the compound represented by Formula 1, 2, 3 or 4 or any mixture thereof.

[0095] Preferably, the electron donor is a cytochrome P450 enzyme reductase. Also preferably, the cytochrome P450 enzyme reductase is selected from the group consisting of Ncp1, ATR1, AnCPR, TaCPR, RnCPR, XlCPR, and BufoCPR.

[0096] Preferably, the catalytic reaction is carried out at 28-30°C for 1-2 days; more preferably, the catalytic reaction is carried out at 30°C for 1 day.

[0097] In the method for preparing bufadienolide compounds, the P450 enzyme can be extracted and purified from the bioengineered bacteria of the present invention, or can be present in the bioengineered bacteria. The extraction and purification can be performed using methods known in the art.

[0098] In a preferred embodiment of the present invention, the P450 enzyme is present in the bioengineered bacteria of the present invention, and the method for preparing the bufadienolide compound comprises the following steps:

[0099] 1) Providing the bioengineered strain of the present invention;

[0100] 2) mixing the bioengineered strain with bufalin and / or resifugin to obtain a mixed culture, and incubating the mixed culture; preferably, the mixed culture further comprises 5-aminolevulinic acid hydrochloride.

[0101] In step 2), the bioengineered strain may be mixed with bufalin to obtain a mixed culture, or the bioengineered strain may be mixed with resinbufogenin to obtain a mixed culture, or the bioengineered strain may be mixed with both bufalin and resinbufogenin to obtain a mixed culture.

[0102] Preferably, in step 2), the OD of the bioengineered strain is 600 The final concentration of bufalin and / or resifugin in the mixed culture is preferably 40-100 μM. Furthermore, the final concentration of bufalin and / or resifugin in the mixed culture is preferably 200 μM to 1 mM, more preferably 200-400 μM. Higher substrate concentrations can result in decreased conversion rates. When the substrates are bufalin and resifugin, the final concentration refers to the combined concentration of bufalin and resifugin.

[0103] Preferably, the mixed culture may further contain 5-aminolevulinic acid hydrochloride. 5-aminolevulinic acid hydrochloride is a precursor for heme synthesis, and heme is a coenzyme for P450 enzymes. When P450 enzymes are overexpressed, heme synthesis in vivo may be insufficient. Therefore, the additional addition of 5-aminolevulinic acid hydrochloride can supplement heme. The final concentration of 5-aminolevulinic acid hydrochloride in the mixed culture is preferably 0.5-1 mM.

[0104] Preferably, the incubation is performed at 28-30° C. and 220-250 rpm for 1-2 days; more preferably, the mixed incubation is performed at 30° C. and 220 rpm for 1 day.

[0105] The incubation can be performed on a thermostatic shaker.

[0106] In some specific embodiments of the present invention, the method for preparing a bufadienolide compound of the present invention comprises the following steps:

[0107] Step (1): dissolving a substrate in a reaction solvent to obtain a substrate solution, wherein the substrate is bufalin and / or resifugin;

[0108] Step (2): mixing the substrate solution obtained in step (1) with the bioengineered strain culture of the present invention at a ratio of 1% (V / V) and incubating the mixture;

[0109] Step (3): The mixture of step (2) is shaken on a shaker for microbial catalysis to obtain a bufadienolide compound as a product, wherein the bufadienolide compound is selected from the compounds represented by the following formulas 1, 2, 3, or 4, or a mixture thereof:

[0110]

[0111] The reaction solvent in the above step (1) can be methanol or N,N-dimethylformamide.

[0112] The method for preparing a bufadienolide compound of the present invention may further include purifying the bufadienolide compound from the resulting reaction product. The purification method may be performed by extraction with ethyl acetate, followed by separation and purification of the desired product from the catalytic reaction product by, for example, preparative liquid chromatography. For example, the compound represented by Formula 1, 2, 3, or 4, or any mixture thereof, may be separated and purified.

[0113] The P450 enzyme CYP46A35 discovered in the present invention can efficiently catalyze the hydroxylation of bufalin and / or resibufogenin under mild conditions to synthesize specifically position-modified bufadienolide compounds. This enzyme exhibits strong regioselectivity and stereoselectivity, specifically catalyzing the hydroxylation of the inert C-H bonds at positions 1 and 19 of bufalin to produce 19-hydroxybufalin and 1β-hydroxybufalin, or specifically catalyzing the hydroxylation of the inert C-H bonds at positions 1 and 2 of resibufogenin to produce 1β-hydroxyresibufogenin and 2β-hydroxyresibufogenin. This invention is efficient, simple, low-cost, environmentally friendly, and has promising industrial application prospects.

[0114] According to another aspect of the present invention, there is also provided the use of the P450 enzyme, the nucleic acid, the expression vector, the enzyme composition and the bioengineering strain of the present invention in the preparation of bufadienolide compounds.

[0115] According to another aspect of the present invention, a bufadienolide compound is provided, which is obtained by catalyzing the P450 enzyme of the present invention with resibufogenin as a substrate in the presence of an electron donor; wherein the structural formula of the bufadienolide compound is shown in Formula 4:

[0116]

[0117] The compound represented by Formula 4 is 2β-hydroxyresibufogenin, a novel, previously unreported compound. The present invention discovered that the compound of Formula 4 (2β-hydroxyresibufogenin) exhibits inhibitory effects on leukemia and lung cancer cells. Its potential applications are worthy of exploration.

[0118] The present invention will be further described in detail below in conjunction with the examples of the present invention. The following examples are illustrative, not restrictive, and the scope of protection of the present invention should not be limited by the following examples. The examples of the present invention are implemented under the premise of the technical solution of the present invention, and detailed implementation schemes and specific operating processes are given, but the scope of protection of the present invention is not limited to the following examples.

[0119] Example

[0120] The experimental methods in the following examples without specifying specific steps and conditions were carried out according to conventional methods and conditions known in the art, or according to the methods and conditions recommended by the manufacturers.

[0121] Unless otherwise specified, the materials, reagents, instruments, etc. used in the examples can be obtained from conventional commercial sources.

[0122] The sources or formulas of the following reagents are as follows:

[0123] The pRS426-HXT7p-FBA1t-Ura3 and pRS425-TEF1p-PGK1t-Leu2 plasmids can be constructed according to the reference "Wang, WF; Xiao, H.; Zhong, JJ, Biosynthesis of a ganoderic acidiin Saccharomyces cerevisiae by expressing a cytochrome P450 gene from Ganodermalucidum. Biotechnol Bioeng 2018, 115(7), 1842-1854"; they can also be constructed by inserting the yeast-derived promoter HXT7p and terminator FBA1t into the pRS426 plasmid, and by inserting the yeast-derived promoter TEF1p and terminator PGK1t into the pRS425 plasmid. Plasmids pRS425 and pRS426 can be purchased from Addgene.

[0124] Bufalin: purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0125] Resifugin: purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0126] Methanol: purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0127] SD-Ura-Leu medium: Purchased from Shanghai Shaoxin Biotechnology Co., Ltd. Add 8 g of powder and dilute to 950 mL. For solid medium, add 20 g of agar powder per liter. Adjust the pH to 6-6.5. Add glucose to a final concentration of 2% before use.

[0128] Ethyl acetate: purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0129] 5-Aminolevulinic acid hydrochloride: purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0130] Saccharomyces cerevisiae BY4742 strain: purchased from Shanghai Baosai Biotechnology Co., Ltd.

[0131] Saccharomyces cerevisiae BJ5464-NpgA strain: purchased from Shanghai Baosai Biotechnology Co., Ltd.

[0132] Saccharomyces cerevisiae YRC01 strain: purchased from Shanghai Baosai Biotechnology Co., Ltd.

[0133] Saccharomyces cerevisiae WAT11U strain: purchased from Shanghai Baosai Biotechnology Co., Ltd.

[0134] Leukemia HL 60 cells: American Type Culture Collection ATCC

[0135] Lung cancer A549 cells: American Type Culture Collection (ATCC)

[0136] Hepatocellular carcinoma HepG2 cells: American Type Culture Collection (ATCC)

[0137] Breast cancer MDA-MB-231 cells: American Type Culture Collection (ATCC)

[0138] Colon cancer SW480 cells: American Type Culture Collection ATCC

[0139] Example 1. Construction of P450 enzyme CYP46A35 expression vector

[0140] 1. RNA was extracted from the postauricular gland of Bufo bufo gargarizans and reverse transcribed to obtain cDNA. Primers CYP46A35-F and CYP46A35-R were designed based on the DNA sequence of CYP46A35 (SEQ ID NO. 2) to amplify the CYP46A35 gene. The CYP46A35 gene fragment was obtained by PCR amplification. Using genomic DNA from Saccharomyces cerevisiae strain BY4742 as a template, primers Ncp1-F and Ncp1-R were used to amplify the cytochrome P450 reductase (CPR) gene ncp1. The CPR genes ATR1 (from Arabidopsis thaliana), AnCPR (from Aspergillus nidulans), and TaCPR (from Trichoderma asperellum) were obtained from other laboratories. Nineday Company synthesized the CPR genes RnCPR from Rattus norvegicus, XlCPR from Xenopus laevis, and BufoCPR from the Chinese toad Bufo bufo gargarizans, and amplified the corresponding fragments using corresponding primers. The nucleotide sequences of the aforementioned CPRs are shown in SEQ ID NOs. 10-16, respectively. The primers used are as follows:

[0141] CYP46A35-F: tttttaattttaatcaaaaaATGGAGCTGCTGGGCCTGG (SEQ ID NO.17)

[0142] CYP46A35-R: atcaattaatttgaattaacgtttTTAGTCAACCTTCTTTCCTTC (SEQ IDNO.18)

[0143] Ncp1-F: atctaagttttaattacaaaatgccgtttggaatagac (SEQ ID NO.19)

[0144] Ncp1-R: cgatttcaattcaattcaatgtttttaccagacatcttcttg (SEQ ID NO.20)

[0145] ATR1-F: atctaagttttaattacaaagtttatgacttctgctttgtatgc (SEQ ID NO. 21)

[0146] ATR1-R:cgatttcaattcaattcaatgttttcaccagacatctctgaggtatc(SEQ ID NO.22)

[0147] AnCPR-F:atctaagttttaattacaaaATGGCACAACTCGATACTC(SEQ ID NO.23)

[0148] AnCPR-R:cgatttcaattcaattcaatgtttTCATGACCAGACATCCTC(SEQ ID NO.24)

[0149] TaCPR-F:atctaagttttaattacaaaATGGCGGAATTGGACACGTTGGAC(SEQ ID NO.25)

[0150] TaCPR-R:cgatttcaattcaattcaatgtttTTATGACCAGACATCCTCCTGG(SEQ ID NO.26)

[0151] RnCPR-F:atctaagttttaattacaaaATGGGTGATTCTCATGAAG(SEQ ID NO.27)

[0152] RnCPR-R:cgatttcaattcaattcaatgtttTTAAGACCAAACATCTAAAG(SEQ ID NO.28)

[0153] XlCPR-F:atctaagttttaattacaaaATGGGTGAATCTTGTACTGAAC(SEQ ID NO.29)

[0154] XlCPR-R:cgatttcaattcaattcaatgtttTTAAGACCAAACATCTTGAG(SEQ ID NO.30)

[0155] BufoCPR-F:atctaagttttaattacaaaATGGAACCATCTGCTTGTGG(SEQ ID NO.31)

[0156] BufoCPR-R:cgatttcaattcaattcaatgtttTTAAGACCAAACATCTTGAG(SEQ ID NO.32)

[0157] 2. Construction of expression vector containing CYP46A35 gene and CPR gene

[0158] The linearized plasmid vectors pRS426-HXT7p-FBA1t-Ura3 and pRS425-TEF1p-PGK1t-Leu2 were obtained by single-restriction digestion with the restriction endonuclease MssI. Using a one-step cloning method, the CPR gene amplified fragment was homologously recombined with the linearized plasmid vector pRS425-TEF1p-PGK1t-Leu2; the CYP46A35 gene was homologously recombined with the linearized plasmid vector pRS426-HXT7p-FBA1t-Ura3. The homologous recombination products were transformed into competent Escherichia coli DH5α cells, and transformants were selected on LB plates containing 100 μg / mL ampicillin. Positive clones were identified by sequencing to obtain recombinant plasmids.

[0159] Example 2. Construction of a bioengineered strain expressing CYP46A35

[0160] The recombinant expression vector obtained in Example 1 was extracted and used to transform competent cells of the Saccharomyces cerevisiae strain WAT11U, which does not express the CYP46A35 enzyme or the cytochrome P450 reductase RnCPR. Transformants were screened using uracil- and leucine-deficient plates to obtain bioengineered strain A.

[0161] The bioengineered strain constructed above was inoculated into SD-Ura-Leu medium and cultured until OD 600 The OD value was about 2, and then 2% was transferred (2% (V / V) access medium) to SD-Ura-Leu medium, cultured for 2 days, and the OD value was about 2. 600 The OD value was about 4.5, and the cells were centrifuged at 5000 g for 5 min. The cells were then concentrated with potassium phosphate buffer (50 mM, pH 7.4) to an OD value of 600 to 40 to obtain a culture of strain A.

[0162] As a control, a recombinant expression vector containing the RnCPR gene was used to transform the Saccharomyces cerevisiae strain WAT11U to construct bioengineered strain B. This strain was transformed only with the recombinant expression vector containing the cytochrome P450 reductase RnCPR, and therefore lacked expression of the P450 enzyme CYP46A35. Separately, Saccharomyces cerevisiae strain WAT11U was transformed with the empty vectors pRS426-HXT7p-FBA1t-Ura3 and pRS425-TEF1p-PGK1t-Leu2, respectively, to construct bioengineered strains C and D. These strains lack expression of the progesterone C21 hydroxylase CYP46A35 and cytochrome P450 reductase. Cultures of strains B, C, and D were obtained using the same method as described above.

[0163] Example 3. Catalytic synthesis of bufadienolide compounds using bufalin as a substrate

[0164] Bufalin was used as a substrate and dissolved in methanol at a bufalin concentration of 20 mM.

[0165] Take the culture of yeast engineered strain A obtained in Example 2, and its OD 600 The concentration of 5-aminolevulinic acid was 40. 5-Aminolevulinic acid hydrochloride was added to a final concentration of 1 mM and bufalin was added to a final concentration of 200 μM, and the culture was incubated at 30° C. and 220 rpm for 1 day using a shaker.

[0166] The biocatalytic system solution was extracted twice with twice the volume of ethyl acetate and centrifuged at 15,000 rpm for 15 minutes. The ethyl acetate layer was evaporated in a rotary concentrator at 1,000 rpm and 30°C. Methanol was added for reconstitution, and the solution was centrifuged at 15,000 rpm for 15 minutes. The supernatant was filtered through a 0.22 μm filter to obtain the product solution.

[0167] The product solution was detected by ultra-high performance liquid chromatography (HPLC). HPLC detection conditions were as follows: Agilent 1260 Infinity II liquid chromatography system, C18 reverse phase column (model: InfinityLab Poroshell 120); mobile phase A: 0.1% formic acid in water, mobile phase B: acetonitrile, gradient elution; column temperature: 30°C, elution rate: 0.5 mL / min, diode array detector (DAD) UV detection wavelength: 300 nm.

[0168] As a control, the culture of the engineered yeast strain B obtained in Example 2 was used to react with bufalin as a substrate using the same method as above and detected by HPLC.

[0169] The HPLC results were as follows Figure 3 As shown, i) is the HPLC curve of the bufalin standard; iii) is the HPLC curve of the product obtained by biocatalysis of the bioengineered strain A (WAT11U (RnCPR+CYP46A35)). The bioengineered strain was transformed with the P450 enzyme CYP46A35 gene and the cytochrome P450 enzyme reductase RnCPR gene, and its reaction product curve showed a new peak different from bufalin. The product components were subjected to high-resolution mass spectrometry and nuclear magnetic resonance (for specific data, see Example 5 and Figure 5 ) verified that the two main new peaks are as follows Figure 3 The product shown contains 19-hydroxybuflin and 1β-hydroxybuflin. Curve iii) shows that the majority of buflin is converted to 1β-hydroxybuflin, with a considerable portion also converted to 19-hydroxybuflin, with 1β-hydroxybuflin and 19-hydroxybuflin being the main products. ii) shows the HPLC curve of the product obtained by biocatalysis using the control bioengineered strain B (WAT11U (RnCPR)), which was transfected with only the cytochrome P450 reductase gene, but not the CYP46A35 gene. The reaction product curve shows the presence of a significant amount of buflin, while no peaks for 19-hydroxybuflin and 1β-hydroxybuflin are observed. This indicates that the bioengineered strain B, which lacks the CYP46A35 gene, is unable to catalyze the site-specific hydroxylation reaction of buflin.

[0170] In addition, as a control, cultures of the engineered yeast strains C and D obtained in Example 2 were reacted with bufalin as a substrate using the same method as above and analyzed by HPLC. It was found that the engineered yeast strains C and D were also unable to catalyze the bioconversion of bufalin.

[0171] The above results also prove that the bioengineered strain constructed by the present invention successfully transcribed and expressed the transferred P450 enzyme CYP46A35 gene and cytochrome P450 enzyme reductase gene, and that both expressed enzymes can exert the desired biological activity and effectively play the role of substrate catalysis and electron transfer.

[0172] Example 4. Catalytic synthesis of bufadienolide compounds using resibufogenin as substrate

[0173] Resifugin as a substrate was dissolved in methanol at a resifugin concentration of 20 mM.

[0174] Take the culture of yeast engineered strain A obtained in Example 2, and its OD 600 The concentration of 5-aminolevulinic acid was 40. 5-Aminolevulinic acid hydrochloride was added to the culture at a final concentration of 1 mM and resibufogenin was added to the culture at a final concentration of 200 μM, and the culture was incubated at 30° C. and 220 rpm for 1 day using a shaker.

[0175] The biocatalytic system solution was extracted twice with twice the volume of ethyl acetate and centrifuged at 15,000 rpm for 15 minutes. The ethyl acetate layer was evaporated in a rotary concentrator at 1,000 rpm and 30°C. Methanol was added for reconstitution, and the solution was centrifuged at 15,000 rpm for 15 minutes. The supernatant was filtered through a 0.22 μm filter to obtain a product solution. The product solution was analyzed by ultra-high performance liquid chromatography (HPLC). The HPLC detection conditions were the same as those in Example 3.

[0176] As a control, the culture of the engineered yeast strain B obtained in Example 2 was used to react with resibufogenin as a substrate using the same method as above and detected by HPLC.

[0177] The HPLC results were as follows Figure 4 As shown, i) is the HPLC curve of the standard product of resifugin; iii) is the HPLC curve of the product obtained by biocatalysis of the bioengineered strain A (WAT11U (RnCPR + CYP46A35)), which is transformed with the P450 enzyme CYP46A35 gene and the cytochrome P450 enzyme reductase gene. The reaction product curve shows a new peak different from resifugin, and the product components are subjected to high-resolution mass spectrometry and nuclear magnetic resonance (for specific data, see Example 5 and Figure 5 ) verification, confirming that the new peak Figure 4 Shown are 1β-hydroxyresibufogenin and 2β-hydroxyresibufogenin. Curve iii) shows that resibufogenin is converted into 1β-hydroxyresibufogenin and 2β-hydroxyresibufogenin. ii) is the HPLC curve of the products obtained by biocatalysis using the control bioengineered strain B (WAT11U (RnCPR)), which only had the cytochrome P450 reductase gene transferred in, without the P450 enzyme CYP46A35 gene. The reverse product curve shows that resibufogenin is still present in large quantities, while no peaks for 1β-hydroxyresibufogenin and 2β-hydroxyresibufogenin are observed. This indicates that bioengineered strain B, which does not have the P450 enzyme CYP46A35 gene transferred in, cannot catalyze the hydroxylation reaction at a specific site of resibufogenin.

[0178] In addition, as a control, cultures of the engineered yeast strains C and D obtained in Example 2 were reacted with resibufogenin as a substrate using the same method as above and analyzed by HPLC. It was found that the engineered yeast strains C and D were also unable to catalyze the bioconversion of resibufogenin.

[0179] The above results also prove that the bioengineered strain constructed by the present invention successfully transcribed and expressed the transferred P450 enzyme CYP46A35 gene and cytochrome P450 enzyme reductase gene, and that both expressed enzymes can exert the desired biological activity and effectively play the role of substrate catalysis and electron transfer.

[0180] Example 5. High-resolution mass spectrometry and nuclear magnetic resonance verification of the product

[0181] The product solution obtained in Example 3 or 4 was separated on a C18 X-bridge (5 μm, 10 × 250 mm) chromatographic column using an Agilent 1260 Infinity II preparative HPLC system (Agilent Technologies, USA). The purity and precise molecular weight of the compound were detected using an Agilent 6120 Quadruple HPLC-HRMS system (Agilent Technologies, USA) under essentially the same conditions as those for HPLC detection of the compound (except that the flow rate of HPLC-HRMS was 0.4 mL / min). The separated high-purity compound was evaporated to dryness, dissolved in deuterated methanol, and subjected to nuclear magnetic resonance (NMR) spectroscopy, and its structure was analyzed based on its molecular weight and nuclear magnetic spectrum.

[0182] The structural analysis of the reaction product of Example 3 is as follows.

[0183] 19-Hydroxybuflin (compound of formula 1):

[0184]

[0185] The high-resolution mass spectrometry calculated value C of the compound of formula 1 24 H 34 O5[M+H] + : 403.2479, high resolution mass spectrometry measured value 403.2484 (such as Figure 5 shown).

[0186] 1 H NMR (600 MHz, methanol-d4): δ 7.98 (dd, J = 9.7, 2.5 Hz, 1H, H23), 7.42 (m, 1H, H21), 6.27 (dd, J = 9.7, 0.6 Hz, 1H, H22), 4.04 (m, 1H, H3), 3.83 (d, J = 11.2 Hz, 1H, H19), 3.40 (s, 2H), 0.70 (s, 3H, H-18).

[0187] 13C NMR (150 MHz, methanol-d4): 164.84 (C24), 150.48 (C21), 149.39 (C22), 125.07 (C20), 115.47 (C23), 86.29 (C14), 67.51 (C3), 66.00 (C19), 52.28 (C17), 49.83 (C13), 42.87 (C 8),42.32(C10),40.56(C12),36.54(C9),34.12(C4),33.00(C5),29.88(C15),29. 72(C2),28.29(C16),27.48(C6),24.23(C1),22.73(C7),22.20(C11),17.38(C18).

[0188] By comparing with the NMR data of reported compounds, the structure of the compound of formula 1 was determined to be 19-hydroxybuflin.

[0189] 1β-Hydroxybuflin (compound of formula 2):

[0190]

[0191] The high-resolution mass spectrometry calculated value C of the compound of formula 2 24 H 34 O5[M+H] + : 403.2479, high resolution mass spectrometry measured value 403.2487 (such as Figure 5 shown).

[0192] 1 H NMR (600MHz, methanol-d4): δ7.99 (dd, J=9.7, 2.5Hz, 1H, H23), 7.41 (m, 1H, H21), 6.27 (dd, J=9.7, 0.5Hz,1H,H22),4.13(brs,1H,H3),3.77(brs,1H,H1),1.08(s,3H,H19),0.72(s,3H,H18).

[0193] 13C NMR (150 MHz, methanol-d4): δ 164.82 (C24), 150.51 (C21), 149.37 (C22), 125.04 (C20), 115.47 (C23), 85.98 (C14), 74.79 (C1), 69.45 (C3), 52.23 (C17), 49.83 (C13), 43.07 (C8), 41.75 (C10), 41.21 (C12), 38.72 (C9), 34.32 (C4), 33.11 (C5), 31.87 (C2), 29.87 (C16), 27.39 (C6), 22.55 (C7), 22.28 (C11), 19.43 (C19), 17.35 (C18).

[0194] By comparing with the NMR data of reported compounds, the structure of the compound of formula 2 was determined to be 1β-hydroxybuflin.

[0195] The structure of the reaction product of Example 4 is analyzed as follows.

[0196] 1β-Hydroxyresiobafuginin (compound of formula 3):

[0197]

[0198] The high-resolution mass spectrometry calculated value C of the compound of formula 3 24 H 32 O5[M+H] + : 401.2323, high resolution mass spectrometry measured value 401.2323 (such as Figure 5 shown).

[0199] 1 H NMR (600MHz, methanol-d4): δ7.89 (dd, J=9.7, 2.3Hz, 1H, H23), 7.44 (d, J=1.9Hz, 1H, H21), 6.26 (dd, J=9.7, 0.8 Hz,1H,H22),4.12(brs,1H,H15),3.79(s,1H,H3),3.60(brs,1H,H1),1.10(s,3H,H19),0.78(s,3H,H19).

[0200] 13C NMR (150 MHz, methanol-d4): δ 164.5 (C24), 151.81 (C21), 149.58 (C22), 124.5 (C20), 15.40 (C23), 75.6 (C14), 74.59 (C1), 69.40 (C3), 61.14 (C15), 46.24 (C17), 42.22 (C13), 41.25 (C9), 39.94 (C10), 35.17 (C12), 34.22 (C4), 33.21 (C8), 33.02 (C6), 31.86 (C2), 26.56 (C6), 22.20 (C7), 21.45 (C11), 19.41 (C19), 17.16 (C18).

[0201] By comparing with the NMR data of reported compounds, the structure of the compound of formula 3 was determined to be 1β-hydroxy bufotoxin.

[0202] 2β-Hydroxyresiobafuginin (compound of formula 4):

[0203]

[0204] The high resolution mass spectrometry calculated value C of the compound of formula 4 24 H 32 O5[M+H] + : 401.2323, high resolution mass spectrometry measured value 401.2336 (such as Figure 5 shown).

[0205] The H-NMR and C-NMR data of the compound of formula 4 are shown in Table 1 below:

[0206] Table 1. H NMR and C NMR data of compound 4

[0207]

[0208]

[0209] a Data measured at 600MHz( 1 H) and 150MHz( 13 C).

[0210] According to the high-resolution mass spectrum m / z 401.2336[M+H] + The pseudo molecular ion peak of the compound was determined to be C 24 H 32 O5, indicating that the compound is a monohydroxylated product of lipofugin. Figure 6 As shown, compared with resifugin, the Dept135 and 13The C spectrum has an additional CH signal at δ71.1. 1 H- 1 H COSY correlations indicated that the hydroxyl group in this compound was introduced at C2. In the NOESY spectrum, enhanced NOEs for H2 and H4 (δ 1.45) indicated that the 2-hydroxyl group was in the β-configuration. Based on these findings, this compound was identified as 2β-hydroxyresiobafuginin.

[0211] Example 6. Optimization of biocatalysis

[0212] The conversion efficiency of bufalin and resifobafoxin catalyzed by different bioengineering strains was studied to screen for high-efficiency bioengineering strains.

[0213] 1. Study on the transformation rate of different chassis strains

[0214] The following bioengineering strains were constructed using the same method as for preparing bioengineering strain A in Example 2, except that the chassis strains were different and transformed with recombinant expression vectors for the CYP46A35 gene and the Ncp1 gene. Biocatalytic reactions were then performed using the same method as in Examples 3 or 4. The results are listed in Tables 2 and 3 below.

[0215] Table 2. Transformation results of bufalin by different strains expressing CYP46A35 and Ncp1

[0216]

[0217] Table 3. Transformation results of resifugin by different strains expressing CYP46A35 and Ncp1

[0218]

[0219]

[0220] Comparing the above results, the WAT11U strain had a relatively high transformation efficiency. Subsequently, the WAT11U strain was used as the base strain to test the transformation of different CPRs.

[0221] 2. Study on the transformation efficiency of different CPRs inserted into the WAT11U strain

[0222] The following bioengineered strains were constructed using the same method as in Example 2, except that recombinant expression vectors for the CYP46A35 gene and different CPR genes were used for transformation. Biocatalytic reactions were then performed using the same method as in Example 3 or 4. The results are listed in Tables 4 and 5 below.

[0223] Table 4. Conversion results of bufalin by WAT11U strain expressing CYP46A35 and different CPRs

[0224]

[0225] Table 5. WAT11U strain expressing CYP46A35 and the results of different CPRs on the conversion of resifugin

[0226]

[0227] The above results indicate that the combination of RnCPR from Rattus norvegicus and CYP46A35 exhibited the highest conversion efficiency, with a total conversion rate of 67.87±0.94% for bufalin and 61.06±0.30% for resifobufogenin. The combination of BufoCPR from the Chinese giant toad (Bufo bufo gargarizans) and CYP46A35 also exhibited high conversion rates, with total conversion rates of 65.08±0.17% for bufalin and 53.49±0.96% for resifobufogenin. While the substrates bufalin and resifobufogenin could be converted to their corresponding hydroxylated products in the absence of an exogenous CPR, the conversion rates were lower.

[0228] Example 7. Determination of antitumor activity of the product

[0229] The inhibitory effects of the compounds of Formulas 1, 2, 3, and 4 prepared by the method of the present invention on different tumor cell lines were studied.

[0230] The specific experimental method is as follows: tumor cells were cultured in RMPI-1640 or DMEM medium supplemented with 10% fetal bovine serum and cultured in a 37°C, 5% CO2 incubator. The cytotoxicity of the compound to tumor cells was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethyloxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) method. Briefly, cells were plated at 5×10 cells per well. 3 Cells were seeded into 96-well plates at a density of 100 cells / mL and incubated in culture medium at 37°C for 12 h. Compounds were added to the cell cultures at different concentrations in triplicate, and cisplatin was used as a positive control. The IC of each compound was calculated using the method of Reed and Muench. 50 The results are shown in Table 6.

[0231] Table 6. IC against five tumor cell lines 50 Value (cisplatin as positive control)

[0232]

[0233] a: Leukemia HL 60 cells

[0234] b: Lung cancer A549 cells

[0235] c: Hepatocellular carcinoma HepG2 cells

[0236] d: breast cancer MDA-MB-231 cells

[0237] e: Colon cancer SW480 cells

[0238] From the above results, it can be seen that the compounds of formula 1 and formula 2 (19-hydroxybuflin and 1β-hydroxybuflin) showed much higher inhibitory effects on these five tumor cells than cisplatin; the compound of formula 1 had an IC of 0. 50 The value is in the nM level, which is comparable to that reported in the literature. As described in the background introduction, the compound can also reduce the weight and volume of tumors in a xenograft lung cancer mouse model. According to the test results of the present invention, the IC value of the compound of formula 1 for leukemia HL 60 cells is 50 The value is also at the nM level, indicating that it also has a strong killing effect on leukemia cells, so it has good application prospects; the IC of the compound of formula 2 on the five tested tumor cells 50 The values ​​are in the range of 16-95 nM, indicating that it has the potential to become a potent broad-spectrum anti-tumor drug; the compound of formula 3 (1β-hydroxylipolytoxin) exhibits an inhibitory effect on leukemia HL 60 cells and lung cancer A549 cells that is significantly higher than that of cisplatin; the compound of formula 4 (2β-hydroxylipolytoxin) has an inhibitory effect on lung cancer A549 cells that is comparable to that of cisplatin.

[0239] The preferred embodiments of the present invention have been described in detail above. It should be understood that numerous modifications and variations based on the concepts of the present invention are possible without inventive effort by those skilled in the art. Therefore, any technical solution that can be derived by one skilled in the art through logical analysis, reasoning, or limited experimentation based on the concepts of the present invention and the prior art should be within the scope of protection defined by the claims.

Claims

1. A P450 enzyme, characterized in that The amino acid sequence of the P450 enzyme is shown in SEQ ID NO.

1.

2. A nucleic acid encoding the P450 enzyme according to claim 1.

3. The nucleic acid according to claim 2, wherein the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO.

2.

4. An expression vector, characterized in that The expression vector comprises a nucleic acid encoding the P450 enzyme according to claim 1. The expression vector according to claim 4 , wherein the expression vector comprises the nucleotide sequence shown in SEQ ID NO.

2.

6. The expression vector according to claim 4, wherein the expression vector comprises the constitutive promoter HXT7p upstream of the nucleic acid encoding the P450 enzyme according to claim 1; preferably, the backbone of the expression vector is pRS426 or pRS425.

7. An enzyme composition comprising the P450 enzyme according to claim 1 and cytochrome P450 enzyme reductase.

8. The enzyme composition of claim 7, wherein the cytochrome P450 enzyme reductase is selected from the group consisting of Ncp1, ATR1, AnCPR, TaCPR, RnCPR, XlCPR, and BufoCPR.

9. A bioengineered strain, characterized in that: The bioengineered strain expresses the P450 enzyme according to claim 1; preferably, the bioengineered strain also expresses a cytochrome P450 enzyme reductase; preferably, the cytochrome P450 enzyme reductase is selected from Ncp1, ATR1, AnCPR, TaCPR, RnCPR, XlCPR and BufoCPR.

10. The bioengineered strain according to claim 9, wherein the bioengineered strain is selected from the group consisting of Saccharomyces cerevisiae strains BY4742, BJ5464-NpgA, YRC01 and WAT11U.

11. A method for preparing a bufadienolide compound, comprising contacting the P450 enzyme according to claim 1 with bufalin and / or resifugin in the presence of an electron donor, thereby performing a catalytic reaction to produce a compound of Formula 1, 2, 3 or 4 or a mixture thereof:

12. The method according to claim 11, wherein the electron donor is a cytochrome P450 enzyme reductase; preferably, the cytochrome P450 enzyme reductase is selected from Ncp1, ATR1, AnCPR, TaCPR, RnCPR, XlCPR and BufoCPR.

13. The method according to claim 11, wherein the catalytic reaction is carried out at 28-30°C for 1-2 days; preferably, the catalytic reaction is carried out at 30°C for 1 day.

14. The method according to claim 11, wherein the P450 enzyme according to claim 1 is contacted with bufalin to perform a catalytic reaction to produce the compounds represented by Formula 1 and Formula 2; or the P450 enzyme according to claim 1 is contacted with resifugin to perform a catalytic reaction to produce the compounds represented by Formula 3 and Formula 4.

15. The method according to claim 11, wherein the method comprises the following steps: 1) Providing the bioengineered strain according to claim 9 or 10; 2) mixing the bioengineered strain with bufalin and / or resifugin to obtain a mixed culture, and incubating the mixed culture; preferably, the mixed culture further comprises 5-aminolevulinic acid hydrochloride.

16. The method according to claim 15, wherein in step 2), the OD of the bioengineering strain is 600 The final concentration of bufalin and / or resifugin in the mixed culture is 200 μM to 1 mM, and the final concentration of 5-aminolevulinic acid hydrochloride in the mixed culture is 0.5-1 mM.

17. The method according to claim 15, wherein the mixing and incubation is carried out at 28-30°C and 220-250 rpm for 1-2 days; preferably, the mixing and incubation is carried out at 30°C and 220 rpm for 1 day.

18. The method according to claim 11, wherein the method further comprises purifying the compound represented by Formula 1, 2, 3 or 4 or a mixture thereof from the system obtained by the catalytic reaction.

19. Use of the P450 enzyme according to claim 1, the nucleic acid according to claim 2 or 3, the expression vector according to any one of claims 4 to 6, the enzyme composition according to claim 7 or 8, or the bioengineered strain according to claim 9 or 10 in preparing the compound of formula 1, 2, 3 or 4 or a mixture thereof.

20. A bufadienolide compound, which is obtained by catalyzing the P450 enzyme according to claim 1 with resibufogenin as a substrate in the presence of an electron donor; wherein: The structural formula of the bufadienolide compound is shown in Formula 4:

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